Detection of DBD-Carbamoyl Amino Acids in Amino Acid Sequence andd/lConfiguration Determination of Peptides with Fluorogenic Edman Reagent 7-[(N,N-Dimethylamino)sulfonyl]-2,1,3- benzoxadiazol-4-yl Isothiocyanate

✍ Scribed by Yong Huang; Hirokazu Matsunaga; Akira Toriba; Tomofumi Santa; Takeshi Fukushima; Kazuhiro Imai

• Publisher: Elsevier Science
• Year: 1999
• Tongue: English
• Weight: 141 KB
• Volume: 270
• Category: Article
• ISSN: 0003-2697
• DOI: 10.1006/abio.1999.4031

✦ Synopsis

A method for amino acid sequence and D/L configuration identification of peptides by using fluorogenic Edman reagent 7-[(N, N-dimethylamino)sulfonyl]-2,1,3-benzoxadiazol-4-yl isothiocyanate (DBD-NCS) has been developed. This method was based on the Edman degradation principle with some modifications. A peptide or protein was coupled with DBD-NCS under basic conditions and then cyclized/cleaved to produce DBD-thiazolinone (TZ) derivative by BF3, a Lewis acid, which could significantly suppress the amino acid racemization. The liberated DBD-TZ amino acid was hydrolyzed to DBD-thiocarbamoyl (TC) amino acid under a weakly acidic condition and then oxidized by NaNO2/H+ to DBD-carbamoyl (CA) amino acid which was a stable and had a strong fluorescence intensity. The individual DBD-CA amino acids were separated on a reversed-phase high-performance liquid chromatography (RP-HPLC) for amino acid sequencing and their enantiomers were resolved on a chiral stationary-phase HPLC for identifying their D/L configurations. Combination of the two HPLC systems, the amino acid sequence and D/L configuration of peptides could be determined. This method will be useful for searching D-amino-acid-containing peptides in animals.